Connecting TDP-43 Pathology to the Molecular Profiles of Neurodegeneration
将 TDP-43 病理学与神经退行性变的分子特征联系起来
基本信息
- 批准号:10057068
- 负责人:
- 金额:$ 260.93万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-08-15 至 2023-07-02
- 项目状态:已结题
- 来源:
- 关键词:3-DimensionalALS patientsAddressAffectAgeAlzheimer&aposs DiseaseAlzheimer&aposs disease patientAstrocytesAtlasesAutopsyCRISPR interferenceCell modelCellsCoculture TechniquesDataDementiaDiseaseFrontotemporal DementiaFunctional disorderGenetic TranscriptionGenomicsGrantHumanImageImmune signalingIndividualInflammatoryLinkMediatingMicrogliaMolecularMolecular ProfilingMotor CortexNerve DegenerationNeurodegenerative DisordersNeurogliaNeuronsOrganoidsOxidative StressPathologicPathologyPathway interactionsPatientsProteinsReportingResolutionRetrotransposonSamplingSignal PathwaySliceSudden DeathSystemTDP-43 aggregationTREM2 geneTestingTissue BanksTissue SampleTissue StainsTissuesToxic effectWorkbasecell typeexperimental studyfrontal lobefrontotemporal lobar dementia-amyotrophic lateral sclerosisglial activationhistological stainsinduced pluripotent stem cellinflammatory markerinnate immune pathwaysneuroinflammationneurotoxicneurotoxicityprofiles in patientsprotein TDP-43protein aggregationpseudotoxoplasmosis syndrometranscriptometranscriptomics
项目摘要
Connecting TDP-43 Pathobiology to the Molecular Profiles of TDP-43 Driven Common Dementias
In previous collaborative work[1], Dr. Phatnani and Dr. Gale-Hammell used a large ALS patient sequencing consortium to uncover the predominant molecular pathways that are altered in the frontal and motor cortex of patients with this TDP-43 associated disease. In this work, ALS cortex samples could be differentiated into 3 distinct groups based upon whether their transcriptional profiles showed hallmarks of: oxidative stress, retrotransposon de-silencing, or neuroinflammation. Histological staining of tissues from these same patients showed tight links between TDP-43 proteinopathy and the latter two groups of patients, though TDP-43 protein aggregates were most predominant in the retrotransposon expressing subset. This grant will build on our prior work to address whether similar TDP-43 dependent pathomechanisms are involved in common dementias associated with TDP-43 dysfunction, specifically frontotemporal dementia with TDP-43 proteinopathy (FTD-TDP) and Alzheimer’s Disease (AD). Our hypothesis is that retrotransposons contribute in part to the toxicity associated with TDP-43 dysfunction in FTD-TDP and AD, which we will conclusively test in this proposal. We have amassed a large collection of tissue samples from over 200 patients with either FTD-TDP or AD, and with extensive clincopathological characterization. The profiles from these samples will be used to guide deep mechanistic studies into the pathomechanisms of TDP-43 in FTD and AD.
Aim 1: How does the cell- and tissue-specific context of TDP-43 proteinopathy affect its impact in FTD and AD? We will use multiplexed immunostaining data of postmortem cortical tissue from 200 FTLD-TDP and AD patients selected to show signatures of TDP-43 pathology, and in age-matched sudden death controls. We will obtain high spatial resolution IHC images of multiple independent factors, including pathological markers (pTDP43 and pTau), markers of cell type, and components of pathways identified to be coincident with TDP-43 dysfunction in our previous work such as inflammatory markers (IBA1, TREM2) and retrotransposon proteins (HERVK-env and L1HS-orf1). At adjacent slices from the same tissue, we will use Spatial Transcriptomics (ST) to define a high-resolution transcriptomic atlas of cell-type specific and context-specific impacts of TDP-43 proteinopathy. These combined molecular profiles will allow us to directly assess the contribution of TDP-43 dysfunction to cell-type specific and proteinopathy-proximal effects.
Aim 2: Do active retrotransposons simply report on TDP-43 dysfunction or contribute to cellular toxicity in FTD and AD? We will obtain long read PacBio sequencing of individual expressed retrotransposons in FTD-TDP and AD patient samples to robustly determine the specific expressed genomic retrotransposon loci in both dementias, and their relative ability to produce functional proteins. We will then use CRISPRa to activate individual retrotransposons identified above in iPS differentiated cortical neurons to test for relative cellular toxicity. In the converse experiment, we will use CRISPRi to inhibit the activity of individual retrotransposons identified above in iPS neurons with TDP-43 dysfunction to test for the relative ability to alleviate TDP-43 mediated neurotoxicity.
Aim 3: Can TDP-43 dependent retrotransposons contribute to activation of astrocytes and microglia? In the NYGC ALS Consortium patients, we previously saw activation of innate immune signaling pathways in samples with evidence of TDP-43 proteinopathy and activated microglia. Moreover, retrotransposons have previously been shown to activate innate immune pathways in other contexts. This aim will establish whether TDP-43 dependent retrotransposons are sufficient to induce activation of adjacent glial cells in cellular models of FTD-TDP and AD. We will express individual retrotransposons in iPS derived cortical neurons and test for the activation of adjacent astrocytes and microglia in a 3D organoid co-culture system. In the converse experiment, we will express TDP-43 dependent retrotransposons in glial cell types, to test whether retrotransposons more potently activate innate immune signaling pathways in astrocytes and microglia, which subsequently secrete neurotoxic factors.
将TDP-43病理生物学与TDP-43驱动的常见痴呆症的分子谱联系起来
在之前的合作工作中[1],Phatnani博士和Gale-Hammell博士使用大型ALS患者测序联盟来揭示这种TDP-43相关疾病患者的额叶和运动皮层中改变的主要分子途径。在这项工作中,ALS皮质样本可以根据其转录谱是否显示氧化应激、逆转录转座子去沉默或神经炎症的标志而分为3个不同的组。来自这些相同患者的组织的组织学染色显示TDP-43蛋白质病与后两组患者之间的紧密联系,尽管TDP-43蛋白质聚集体在逆转录转座子表达亚群中最主要。这项资助将建立在我们之前的工作基础上,以解决类似的TDP-43依赖性病理机制是否涉及与TDP-43功能障碍相关的常见痴呆症,特别是额颞叶痴呆与TDP-43蛋白质病(FTD-TDP)和阿尔茨海默病(AD)。我们的假设是,反转录转座子有助于部分毒性与TDP-43功能障碍FTD-TDP和AD,我们将最终测试在这个建议。我们收集了200多例FTD-TDP或AD患者的大量组织样本,并进行了广泛的临床病理学表征。这些样品的特征将用于指导TDP-43在FTD和AD中的病理机制的深入机制研究。
目的1:TDP-43蛋白病的细胞和组织特异性背景如何影响其在FTD和AD中的作用?我们将使用来自200名FTLD-TDP和AD患者的死后皮质组织的多重免疫染色数据,以显示TDP-43病理学的特征,以及年龄匹配的猝死对照。我们将获得多个独立因素的高空间分辨率IHC图像,包括病理标志物(pTDP 43和pTau),细胞类型标志物,以及在我们以前的工作中被鉴定为与TDP-43功能障碍一致的途径组分,如炎症标志物(IBA 1,TREM 2)和逆转录转座子蛋白(HERVK-env和L1 HS-orf 1)。在来自相同组织的相邻切片上,我们将使用空间转录组学(ST)来定义TDP-43蛋白质病的细胞类型特异性和背景特异性影响的高分辨率转录组图谱。这些组合的分子特征将使我们能够直接评估TDP-43功能障碍对细胞类型特异性和蛋白质病变近端效应的贡献。
目的2:在FTD和AD中,活性反转录转座子是否仅报告TDP-43功能障碍或导致细胞毒性?我们将获得FTD-TDP和AD患者样本中单个表达的逆转录转座子的长读PacBio测序,以稳健地确定两种痴呆症中特异性表达的基因组逆转录转座子位点及其产生功能蛋白的相对能力。然后,我们将使用CRISPRa激活iPS分化的皮质神经元中上述鉴定的单个逆转录转座子,以测试相对细胞毒性。在匡威的实验中,我们将使用CRISPRi来抑制上述在具有TDP-43功能障碍的iPS神经元中鉴定的单个反转录转座子的活性,以测试减轻TDP-43介导的神经毒性的相对能力。
目的3:TDP-43依赖性反转录转座子是否有助于星形胶质细胞和小胶质细胞的活化?在NYGC ALS联盟的患者中,我们先前在具有TDP-43蛋白质病和活化的小胶质细胞证据的样本中看到先天免疫信号传导通路的活化。此外,逆转录转座子先前已被证明在其他情况下活化先天免疫通路。该目的将确定TDP-43依赖性逆转录转座子是否足以诱导FTD-TDP和AD细胞模型中相邻胶质细胞的活化。我们将在iPS衍生的皮质神经元中表达单个反转录转座子,并在3D类器官共培养系统中测试相邻星形胶质细胞和小胶质细胞的活化。在匡威的实验中,我们将在神经胶质细胞类型中表达TDP-43依赖性逆转录转座子,以测试逆转录转座子是否更有效地激活星形胶质细胞和小胶质细胞中的先天免疫信号传导途径,其随后分泌神经毒性因子。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Molly Gale Hammell其他文献
Molly Gale Hammell的其他文献
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A systematic analysis of mir-34 function in C. elegans
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$ 260.93万 - 项目类别:
A systematic analysis of mir-34 function in C. elegans
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7615289 - 财政年份:2009
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$ 260.93万 - 项目类别:
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